CN102038941A - Application of protective antigen Omp25c to preparation of brucellosis vaccine - Google Patents

Abstract

The invention discloses protective antigen Omp25c protein of brucellosis and application thereof to preparation of a brucellosis vaccine. The test result shows that compared with a wild type vaccine strain, an omp25c mutant strain has reduced toxicity, so an omp25c gene plays a role in the toxicity of the vaccine strain. The immune level and the immune protection effect of body fluid and cells induced by an omp25c deletion strain are reduced, so the omp25c promotes the immune response and the immune protection effect of the vaccine strain. Accordingly, the omp25c protein is an important protective antigen of the brucellosis and can enhance the vaccine strain-induced immune response and the immune protection effect. The brucellosis vaccine is prepared from the omp25c protein serving as an active ingredient, so the immune protection effect of the vaccine can be improved.

Description

The application of protective antigen Omp25c in the brucella vaccine preparation

Technical field

The present invention relates to proteic new purposes, particularly relate to the application of important immune protective antigen Omp25c albumen in the preparation brucella vaccine of brucella vaccine strain M5.

Background technology

Brucellosis (brucellosis is called for short brucellosis) is a class zoonosis that is caused by brucella (Brucella), is a kind of important zoonosis, worldwide is widely current.Brucella can infect the mankind, multiple domestic animal and wild animal, patient's cardinal symptom is chronic arthritis and nerve injury, ill domestic animal mainly show as miscarriage and sterile (Shang Deqiu. the brucellosis progress. sick magazine 2004, the 19:204-212 of preventing and treating of place of china; Corbel, M.J.Brucellosis:an overview.Emerg Infect Dis 1997,3:213-21.).Brucellosis is produced not only for China's animal husbandry and is caused enormous economic loss, also the serious threat people's healthy living.In addition, brucella also is used as biological weapons, and national security is existed serious threat.The sickness rate of brucellosis is in rising trend in recent years, and the sickness rate of China's brucellosis is top all previous records, causes the great attention of relevant departments.

Brucellar outer membrane protein is early than being found in early days the eighties in 20th century, outer membrane protein in the structure of stable bacterial adventitia, adapt in born of the same parents' internal and external environment and the opposing born of the same parents aspect such as bactericidal mechanism and play an important role, closely related with brucellar virulence, also be important source (the Schurig G G of immunogenic protein simultaneously, Sriranganathan N, Corbel M J.Brucellosis vaccines:past, present and future.Vet Microbiol 2002,90:479-496.).Therefore, a lot of researchs about brucella mechanism of causing a disease and immune response mechanism all concentrate in the research of outer membrane protein.Joseph P.Connolly etc. are when the immune protein group research of carrying out Brucella abortus, find that Omp25c is the immunogenic protein (Connolly, J.P.et al.Proteomicanalysis of Brucella abortus cell envelope and identification of immunogeniccandidate proteins for vaccine development.Proteomics 2006.6:3767-80.) of Brucella abortus.But be not that all immunogenic proteins all have immune protective, as Omp31, immunogenic proteins such as Bp26 just do not have immune protective.The inventor also finds in the Brucella melitensis external membrane protein group research in early stage; Omp25c albumen is the main member of Brucella melitensis outer membrane protein; but this albumen whether play an important role aspect Brucella melitensis virulence and the immune protective still unclear; the inventor is by disappearance omp25c in vaccine strain and analyze the change of its virulence and immune protective, inquires into the effect of omp25c albumen in brucella vaccine.

Summary of the invention

The object of the present invention is to provide the proteic a kind of new medical usage of Omp25c.

The inventor studies show that, Omp25c albumen plays an important role in the protectiveness of brucella vaccine, has the effect that strengthens the brucella immune protective, can be used widely in the preparation brucella vaccine.

But the active ingredient of brucella vaccine provided by the present invention is above-mentioned excitating organism produces immunity to brucella a protective antigen Omp25c albumen.

The application of immunogenic protein Omp25c in the immune protective albumen of preparation Brucella antibody also belongs to the scope that comprises of the present invention.

Immune protective antigen provided by the invention can be expressed at the brucella vaccine plant height, thereby further improves the immune protective effect of vaccine.

The invention provides the proteic new purposes of a kind of Omp25c, i.e. application in the preparation brucella vaccine.For analyzing Omp25c to the virulence of brucella vaccine strain M5 and the influence of immune protective; the present invention has at first expressed Omp25c albumen; confirm their immunogenicity; make up the omp25c gene delection mutant of M5 then; relatively the virulence and the immune protective of deletion mutation strain and vaccine strain are analyzed the proteic immune protective of Omp25c.Compare with M5, the time-to-live of omp25c mutant in mouse spleen is shorter, fails to be detected when the 4th week.The antibody horizontal testing result shows, omp25c mutant and M5 vaccine strain all can inducing producing specificity antibody in Balb/c mice body, but compare with M5, the humoral immunoresponse(HI) that mutant causes relatively a little less than, the persistent period is shorter.And mutant induces the level of the cytokine IL-10 that produces the reflection humoral immunoresponse(HI) also low more than M5.The mouse spleen lymphocyte of mutant and M5 vaccine strain is the energy secretion of gamma-IFN all, but mutant IFN-γ concentration is starkly lower than vaccine strain.Immunne response result shows that the body fluid of mutant and cellullar immunologic response ability obviously descend reduced immunogenicity.The counteracting toxic substances experimental result shows, the mice that has inoculated the omp25c mutant with the 16M counteracting toxic substances after, immune protective effect descends, and has significant gap with the M5 vaccine strain.Experimental result shows that M5 compares with vaccine strain, and omp25c mutant virulence attenuation of illustrates that the omp25c gene brings into play certain effect in the virulence of M5 vaccine strain.The disappearance of omp25c makes inductive body fluid of M5 and cellular immune level and immune protective effect descend, and illustrates that omp25c has certain influence to immunne response and the immune protective effect of vaccine strain M5.This shows that outer membrane protein Omp25c is the crucial composition of brucella vaccine playing an important role aspect the virulence of brucella vaccine strain M5 and the immune protective, can it prepare brucella vaccine for active ingredient.

Below in conjunction with specific embodiment the present invention is described in further details.

Description of drawings

Fig. 1 is the SDS-PAGE testing result of recombiant protein Omp25c

Fig. 2 is the immunogenicity analysis result of recombiant protein Omp25c

Fig. 3 is the 1% agarose gel electrophoresis testing result that contains the recombiant plasmid pUC19K-NC of sudden change box N-K-C

Fig. 4 is the PCR qualification result of omp25c deletion mutation strain

Fig. 5 is the survival ability testing result of omp25c deletion mutation strain in mouse spleen

Fig. 6 is mice serum total IgG horizontal detection result

Fig. 7 is an IFN-γ testing result

Fig. 8 is the IL-10 testing result

Fig. 9 is the immunoprotection experimental result

The specific embodiment

Method therefor is conventional method if no special instructions among the following embodiment.Among the present invention, Omp25c represents albumen, and italic small letter omp25c represents gene.

Proteic expression of embodiment 1, Omp25c and immunogenicity analysis

One, the proteic expression of Omp25c

1, expresses primer design

B.melitensis 16M has finished genome sequencing, according to the 16M sequence of including among the GenBank and annotation information (GenBank number: AE008918), with the signal peptide sequence of SignalP software analysis gene and remove corresponding signal peptide sequence, behind proteic antigenicity of DNAStar software analysis and the hydrophobicity, design the proteic specific expressed primer of Omp25c with Primer Premier 5 (PREMIERBiosoft International, Palo Alto): BMEI1829-E-F:3 '-ACGTGGATCCGACGCCGTCATTGAACAGG-5 ' and BMEI1829-E-R:3 '-AGCTAAGCTTGAACTTGTAAGCGACACCG-5 '.Polyclone enzyme action site information according to expression vector pET32a adds BamH I restriction enzyme site at 5 ' end of forward primer again, at 5 ' end interpolation Hind III restriction enzyme site of downstream primer.

2, the structure of recombinant expression carrier

With the 16M genome is template, uses above-mentioned primer BMEI1829-E-F and the BMEI1829-E-R method amplification omp25c gene with PCR.The PCR reaction system is: 10 * PCR buffer, 5 μ l, dNTP (2.5mM) 4 μ l, forward primer (20 μ M) 0.5 μ l, downstream primer (20 μ M) 0.5ul, rTaq enzyme (5U/ μ l) 0.25 μ l, template DNA (10ng/ μ l) 1 μ l adds the water polishing to final volume 50 μ l.The PCR reaction condition is: 94 ℃ of 5min; 95 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 60s, totally 30 circulations; 72 ℃ of 10min.After reaction finishes, reclaim pcr amplification product, it is carried out double digestion digestion with BamHI and HindIII, and link to each other with pET32a carrier (available from Takara company) through same enzyme action digestion process, to connect capable the duplicating of product transformed into escherichia coli DH5 α competent cell, be applied on the LB culture medium flat plate that contains the 0.1mg/mL ampicillin 37 ℃ of overnight incubation.The monoclonal that grows send order-checking company to check order after PCR and enzyme action evaluation correctly, and the result obtains sequence and all correct recombinant expression carrier of on position, called after pET32a-25c.

3, the abduction delivering of recombiant protein

Among the recombinant expression carrier pET32a-25c transformed into escherichia coli ER2566 that order-checking is correct, with the resistance LB agar plate screening positive clone that contains the 0.1mg/mL ampicillin.PCR is identified that the expression cloning be positive is seeded in the LB fluid medium that contains the 0.1mg/mL ampicillin, induce, establish negative control simultaneously, the proteic expression of usefulness 12%SDS-PAGE testing goal with the IPTG of 0.5mmol/L.Positive strain is induced in a large number, after the ultrasonic degradation fragmentation, collects respectively and goes up cleer and peaceful precipitation, carries out SDS-PAGE, and fusion rotein is carried out soluble analysis.The SDS-PAGE testing result is (M:Protein Ruler III as shown in Figure 1; 1: negative control; 2:Omp25c (42.44kD)), omp25c can give expression in escherichia coli ER2566 and expect the albumen that size conforms to (42.44kD).After the positive strain of recombiant protein was induced in a large number, centrifugal collection thalline suspended with PBS, and the ultrasonic degradation fragmentation is collected respectively and gone up cleer and peaceful precipitation, carries out purification.

Two, the immunogenicity analysis of recombiant protein Omp25c

Analyze the immunogenicity of recombiant protein Omp25c with western blotting.Method is: with protein expressioning product behind the 12%SDS-PAGE electrophoresis, change film to nitrocellulose filter by wet commentaries on classics method, spend the night with the TBST sealing that contains 5% defatted milk powder, after the TBST rinsing, with the anti-Brucella antibody of the rabbit of purification is one anti-, establish normal rabbit serum simultaneously in contrast, behind the incubated at room 1h, rinsing, the goat-anti rabbit two anti-(Microbiology and Epidemic Disease Inst., Academy of Military-Medical Sciences (C) that adds the horseradish peroxidase-labeled of dilution in 1: 1000, incubated at room 1h immerses film in the DAB substrate solution after the rinsing, cessation reaction when clear to the purpose band.The result is (M:Protein Ruler III as shown in Figure 2; 1:Omp25c), Omp25c albumen can combine with Brucella melitensis serum, has immunogenicity.

The structure of embodiment 2, omp25c mutant

One, design of primers

According to the ORF of omp25c gene (BMEI1829) and the sequence of both sides, the primer of design amplification N end and C end homology arm, N end homology arm amplimer is: BMEI1829-N-F and BMEI1829-N-R, 5 ' end restriction enzyme site is respectively KpnI and Xho I; C end homology arm amplimer is: BMEI1829-C-F and BMEI1829-C-R, 5 ' end restriction enzyme site is respectively Sal I and Hind III.In addition, identify primer (Puc19K-F and BMEI1829-I-R) in the inside of that gene of card and the outside design mutant of target gene homology arm respectively, be used for determining the correct replacement of omp25c gene.The primer and the sequence thereof of design see Table 1.

Table 1 primer sequence

Two, the structure of omp25c sudden change box and mutational vector

With the 16M genome is template, the N end homology arm of omp25c gene at first increases, homology arm N end fragment with amplification is inserted into pUC19K (Qiao Feng earlier, Chen Zeliang, Wang Yufei, structure and the application in the strain of brucella disappearance labelling makes up thereof Deng the .pUC19K plasmid. Chinese biological engineering magazine 2007, Kpn I 27:1-5.) and XhoI site obtain plasmid pUC19K-N; The C that increases then holds homology arm, and the C end fragment is inserted into Sal I and the Hind III site of pUC19K-N, obtains containing the mutational vector pUC19K-NC of sudden change box N-K-C.For further confirming the correct insertion of N, C end homology arm, with Kpn I and Hind III double digestion mutational vector pUC19K-NC is identified respectively, cut out sudden change box N-K-C fragment, 1% agarose gel electrophoresis testing result is (1:pUC19K-NC/KpnI+HindIII as shown in Figure 3; The 2:pUC19K-NC plasmid; M:MarkerIII), consistent with the expection size.With identifying that correct plasmid pUC19K-NC transformed into escherichia coli DH5 α competent cell duplicates, be applied on the LB culture medium flat plate that contains the 0.1mg/mL ampicillin 37 ℃ of overnight incubation.With the monoclonal that grows through PCR and enzyme action identify correct after, send the order-checking of order-checking company, the result obtains sequence and all correct recombinant expression carrier pUC19K-NC of on position.

Three, the screening of omp25c deletion mutant and evaluation

The carrier pUC19K-NC plasmid DNA electricity that will contain the box that suddenlys change is transformed in the brucella M5 electroreception attitude cell, and the coating of recovery back contains the tryptone soya agar TSA flat board (Biomerieux SA) of 50 μ g/mL kanamycin, cultivates 3-5 days screening Amp for 37 ℃ ^sKan ^rResistance clone.Evaluation primer (BMEI1829-I-R and Puc19K-F, sequence sees Table 1) antagonism clone with mutant carries out bacterium colony PCR evaluation, and the result is (M:marker III as shown in Figure 4; 1:M5 Δ omp25c; 2.M5), the clone of picking can amplify the big or small product (1.68Kb) of expection with the evaluation primer, and the wild strain amplification is negative, shows that tentatively it is correct that the deletion mutation strain makes up.To the affirmation of checking order of the resistance clone of the segmental product that can amplify expection size, the result obtains omp25c gene mutation body recombinant bacterial strain, called after M5 Δ omp25c.

Embodiment 3, zoopery

One, laboratory animal and animal immune

Select 6～8 all Balb/c mices in age (Military Medical Science Institute's animal center) for use, be divided into 4 groups at random, 30 every group.The 1st group is M5 attenuated live vaccine (Nat'l Pharmaceutical ﹠ Biological Products Control Institute); The 2nd group is mutant M5 Δ omp25c immune group, immunizing dose 2 * 10 ⁵CFU/ only; The 3rd group is physiology saline control group, and immunizing dose 200 μ L/ only.Take peritoneal injection, immune time is 1 time.

Two, the survival ability of omp25c deletion mutation strain in mouse spleen detects

Use vaccine strain M5 and omp25c deletion mutation strain M5 Δ omp25c immune mouse respectively, the spleen of the 7th, 14 and 28 day aseptic taking-up mice after immunity, each time point is chosen 5 mices for every group, eyeball is put to death mice after getting blood, takes out spleen rapidly, adds the normal saline that 1mL contains 0.1%Triton X-100, make homogenate with glass homogenizer, get tissue homogenate and carry out 10 times of serial dilutions, choose suitable dilution homogenate 100 μ L and be coated with the TSA flat board, calculate CFU.The result as shown in Figure 5, at each time point, the quantity of deletion mutation strain all is less than the positive group of M5 in the M5 Δ omp25c experimental group spleen.In the time of the 28th day, the log value of the bacterial population of M5 is 1.2, and mutant fails to be detected.After this experiment showed the omp25c disappearance, the intracellular bacteria number of brucella M5 reduced, virulence attenuation of.

Three, mice serum total IgG horizontal detection

Respectively at the 7th, 14,28,42,56 day detection mice serum antibody after the immunity, method is: in the mouse tail blood sampling, 4 ℃ are spent the night, the centrifugal 10min of 4000rpm, and separation of serum is stand-by.Adopt indirect non-competing ELISA method to detect the serum antibody level.Concrete operations are with 10 ⁸By elisa plate, by dilution in 1: 400, every hole 100 μ L added sheep anti-mouse igg-HRP reaction to CFU/mL M5 inactivated bacteria, add substrate TMB colour developing at last, place microplate reader to survey 450nm absorbance (OD) value with test serum with every hole 100 μ L bag.The result as shown in Figure 6, M5 group just can obviously detect antibody at the 14th day, antibody titer reached the highest (OD in the 28th day ₄₅₀=0.845),, still still maintains higher level though the 42nd, 56 day antibody horizontal slightly descends.Though M5 Δ omp25c experimental group can detect antibody at the 14th day, be not as remarkable as the positive group of M5, antibody titer reached the highest (OD at the 28th day ₄₅₀Begin during=0.435), by the 42nd day to descend.Though antibody titer variation tendency and the M5 of M5 Δ omp25c are similar, the antibody horizontal of its each time point all is starkly lower than M5 attenuated live vaccine group (P＜0.05).

Four, cytokines measurement

1, the separation of mouse boosting cell

Different time points after immunity is respectively got 3 mices and is put to death and the aseptic spleen of getting from each experimental group, isolate unicellularly with 200 order copper mesh, splenocyte is added in the centrifuge tube abandon supernatant behind the centrifugal 15min of 1000rpm/min.With hypotonic erythrocyte cracked liquid room temperature lucifuge splitting erythrocyte 15min, the centrifugal supernatant of abandoning.1640 culture medium that contain 2% calf serum with 30mL are washed cell 2 times again, containing 1640 complete medium re-suspended cells of 10% calf serum, and counting, the cell concentration dilution is 5 * 10 the most at last ⁶/ mL.

2, stimulated in vitro

100 μ L adding Tissue Culture Plate (5 * 10 is got in immunity and control mice splenocyte respectively ⁵Cells/well), different strains is got 100 μ L and is added corresponding hole, every hole 10 ⁸CFU, every kind of bacterial strain repeats 3 holes.Respectively with ConA (concentration is 5 μ g/mL, Sigma company) and the positive and negative control of 1640 complete mediums of 100 μ L.Mixture is cultivated at 37 ℃, saturated humidity and 5%CO ₂Incubator in, get supernatant behind the 72h and detect IFN-γ and IL10.The test kit of surveying IFN-γ/IL10 adopts R ﹠amp; The Mouse IFN-γ of D System company/IL-10 Immunoassay Kit, the cell conditioned medium sample that dilution is good joins in the enzyme linked plate holes, uses the double antibodies sandwich method, and its OD is surveyed in the reaction back ₄₅₀Value.

3, IFN-γ detects

Brucella is a kind of born of the same parents' endophyte, and the meaning of cellular immunization is greater than humoral immunization.Therefore the present invention has detected the level of mice serum cytokines IFN-γ in M5 and M5 Δ omp25c immunologic process, and the result as shown in Figure 7.Compare with the PBS matched group, the M5 bacterial strain promptly excited cellular immunization to a certain degree in back 7 days in immunity, and IFN-γ level continue to rise subsequently, and had reached peak 42 days of monitoring, and after immunity the 56th day, concentration also maintained higher level.M5 Δ omp25c group has been induced the IFN-γ of certain level in the time of 14 days, reach summit in the time of 28 days, but IFN-γ level has begun to descend in the time of 42 days.Experimental result shows that after the omp25c disappearance, brucella induces the level that produces IFN-γ obviously to reduce.Illustrate that Omp25c albumen brought into play important effect in the cellular immunization of brucella M5.

4, IL-10 detects

According to the literature, IL-10 mainly is the certain effect of performance in brucellar humoral immunization.Therefore the present invention has detected the level of mice serum cytokines IL-10 in M5 and M5 Δ omp25c immunologic process, and the result as shown in Figure 8.Compare with the PBS matched group, M5 and M5 Δ omp25c all can induce the IL-10 of certain level, and variation tendency is roughly approaching.Reach summit in the time of back 42 days in immunity, then descend.But generally, the level of M5 Δ omp25c group IL-10 is lower than the M5 group.In conjunction with mice serum total IgG horizontal detection, illustrate that Omp25c albumen also brought into play important effect in the humoral immunization of brucella M5.

Five, immunoprotection experiment

Behind mouse immune, carry out the counteracting toxic substances experiment the 8th week, intraperitoneal injection virulent strain 16M (Nat'l Pharmaceutical ﹠ Biological Products Control Institute), 10 ⁵Cfu/ only.The 1st, 2,4 weeks were put to death mice behind the counteracting toxic substances, and the aseptic spleen of getting grinds, and is inoculated in the TSA flat board after the dilution, cultivates colony counting, log 3～4 days ₁₀The meansigma methods of CFU ± SD is every group counteracting toxic substances result.The result as shown in Figure 9, behind brucella 16M virulent strain counteracting toxic substances, with the PBS group relatively, M5 Δ omp25c does not produce significant protection power, and than the immune protective effect significant difference (P＜0.05) of M5 attenuated live vaccine.The result shows that the immune protective effect of omp25c deletion mutation strain obviously descends, and illustrates that once more Omp25c albumen has certain immune protective to brucella M5.

Brucella is the entozoic tiny spherical bacillus of a kind of born of the same parents, and extremely strong survival ability is arranged in born of the same parents, and in a single day the infection that makes brucella cause takes place, and is difficult to thoroughly cure.Outer membrane protein and brucellar virulence are closely related, also are the important sources of immunogenic protein simultaneously.But the effect in the virulence of strain and vaccine strain and immune protective, brought into play of nobody research Omp25c albumen still.In the above-described embodiments, we have selected the most frequently used Brucella melitensis attenuated live vaccine M5 of China as target, and M5 is carried out the gene mutation transformation, have made up the deletion mutation strain of brucella outer membrane protein omp25c gene.Inquired into virulence, immunogenicity and the protectiveness of deletion mutation strain, in vaccine strain M5 virulence and immune protective, brought into play how effect thereby analyze Omp25c albumen.

In the above-described embodiments, we have at first expressed Omp25c albumen, have analyzed their antigenicity.The result show Omp25c albumen can with the combination of Brucella melitensis serum, have immunogenicity.Then, we have further analyzed Omp25c albumen to the virulence of M5 vaccine strain and the influence of immune protective.Zoopery is the result show, Omp25c albumen is relevant with brucellar virulence, and the deletion mutation strain that lacks this gene obviously weakens the virulence of mice, can not be in the mice body long-term surviving.The antibody horizontal testing result shows, omp25c mutant and vaccine strain M5 all can inducing producing specificity antibody in Balb/c mice body, but compare with M5, the humoral immunoresponse(HI) that mutant causes relatively a little less than, the persistent period is shorter.And mutant induces the level of the cytokine IL-10 that produces the reflection humoral immunoresponse(HI) also low more than M5.Parasitize in the macrophage behind the brucella infection host, therefore the immunity of organism that causes should be based on cellular immunization.It is brucella challenging antigen presenting cell; cause that the Th cell differentiation is Th1 cell (Mosmann T R; Sad S.The expanding universe of T-cell subset:Th1; Th2 and more.Immunol Today; 1996; 17:138-146); thereby Th1 emiocytosis IFN-γ raises the phagocytosis of macrophage; the macrophage that can also directly kill simultaneously the brucella infection reaches protection host's purpose (Murphy A; Sathiyaseelan J; Parent MA; et al.Interferon-gamma is crucial for survivinga Brucella abortus infection in both resistant C57B/6 and susceptible Balb/cmice.Immunology 2003,19:218-220).Therefore, effectively brucella vaccine must to induce with generation IFN-γ be the Th1 type immunoreation of feature.Behind mutant and the vaccine strain M5 immune mouse, mouse spleen lymphocyte is the energy secretion of gamma-IFN all, but mutant IFN-γ concentration is starkly lower than vaccine strain.Immunne response result shows, the body fluid of mutant and cellullar immunologic response ability obviously descend, reduced immunogenicity illustrates that Omp25c has brought into play important effect in the humoral immunization of M5 vaccine and cellular immunization process, can strengthen the cell and the humoral immunization of vaccine.In addition, from the counteracting toxic substances result, the mutant that has lacked omp25c has been lost excellent protection, has significant gap with vaccine strain M5, shows that omp25c has certain influence to the immune protective effect of attenuated live vaccine M5.This shows that outer membrane protein Omp25c can strengthen the immanoprotection action of vaccine playing an important role aspect the virulence of brucella vaccine strain M5 and the immune protective, can it prepare brucella vaccine for active ingredient.The preparation that utilizes outer membrane protein Omp25c to carry out brucella vaccine can be adopted existing conventional method, and this paper will not give unnecessary details.

Claims (2)

1.Omp25c the application of albumen in the preparation brucella vaccine.

2. application according to claim 1 is characterized in that: be the application of immunogenic protein Omp25c in the immune protective albumen of preparation Brucella antibody.

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